Question Answer

Fixation is the... stabilization of proteins in tissue

Stabilization of proteins is done in
order to prevent
tissue destruction during subsequent steps in processing
stabilization is accomplished by denaturing of proteins
The most important step in the
histology laboratory
fixation
Intent of fixation is:
preserve tissue in life-like state;prevent breakdown of cellular
components; coagulate/precipitate proteins (insoluble);keep
from losing proteins during processing
a good fixative will...
penetrate tissue quickly +harden the tissue retain micro
anatomical structure; maintain histological relationships so
the cells can be studied
Autolysis dissolving of cells by enzyme action
Putrefaction
breakdown of tissue by bacterial action from within. also
called postmortem decomposition
T or F: Fixation brings out the
differences in refractive indexes of
tissues.
True
The cell is composed of
proteins, carbohydrates and lipids held loosely together by
hydrogen bonds
Fixation changes the weak hydrogen
bonds into
stable bonded complexes
Fixation causes ________ linkage of
protein molecules.
cross linkage
Cross links makes tissue suitable for
microtomy by...
strengthening and hardening the tissue
RNA and DNA are entrapped in the
nucleus by the process of ...
fixation
Coagulation/Precipitation render the
________ more resilient.
nucleus.Giving it a sharper more intact appearance.
T or F: Lipids in the cell are easily
fixed.
False
Only 2 chemicals can fix lipids so they
are not lost in subsequent processing.
(1) Osmium tetroxide (2) Chromic Acid
Some _________ are usually LOST
during fixation.
carbohydrates
A ________ - storage form of
_________ is retained during fixation.
A glycogen-storage form of glucose is retained during
fixation. This is due to the entrapment by fixed proteins.




Nine factors which influence tissue
fixation:
Temperature, Size, Tissue/Fixative Volume ratio, Time,
Choice of Fixative, Penetration Capability, Tissue storage,
pH, Osmolality
Osmolality is defined as... the #of particles in a solution
normal osmolality isotonic
hypotonic solution will cause tissue to swell (ie. acetic acid)
hypertonic solution will cause tissue to shrink (ie. picric acid)
Fixatives in order of decreasing speed
of penetration:
formaldehyde-acetic acid-mercuric chloride-methy alcohol-
osmium tetroxide-picric acid
The rate of penetration is affected by
_______.
heat
The rate of penetration is NOT
affected by _______.
the concentration of the fixative
NON-coagulant fixatives produce a gel
that makes penetration
difficult.Examples are...
formaldehyde, glutaraldehyde, acetic acid, potassium
dichromate, osmium tetroxide
Coagulant fixatives establish a
network in tissue allowing penetration.
Examples are...
Mercuric chloride; zinc salts; chromium trioxide; picric acid;
and POTASSIUM DICHROMATE AT PH >3.5
Biopsy specimen which cannot be
immediately placed in fixative should
be.
placed in a saline dampened gauze, put in plastic container
and put on ice for the short holding time.
Kidney biopsy specimens for
immunofluorescence frequently are
held, or even mailed in ________.
Michel transport solution.
Acetic Acid is a non-coagulant and...
Does NOT fix or destroy carbohydrates. Does NOT fix lipids.
YES penetrates slowly. YES preserves nucleoproteins.
Acetic acid lyses _________, and thus
their preservation is poor in in any
fixative containing it.
red blood cells
Acetic acid warnings:
add TO water; use under a hood; causes severe burns;
exposure limit of 10ppm
Formaldehyde is colorless ________. gas
Formaldehyde (manufacturer label) =
Formalin (37%-40% in water)
37%-40% formalin
Let's look at Stains...momentarily

2 categories of dyes... Natural & Artificial
Natural dyes are from natural resources (ie. HEMATOXYLIN)
Artificial dyes are from chemical reactions (greatly outnumber natural dyes)




Biological Stain Commission agency responsible for ensuring the quality of biological
stains and -PROMOTE COOPERATION AMONG VENDORS
-EDUCATE USERS OF STAINS PUBLISH INFO
CONCERNING NEW OR IMPROVED STAINS
Biological stain commission DOES
NOT
manufacture biological stains
Dyes are substances...
capable of imparting color chemically or physically binding
with materials
Dyes impart color due to
the presence of color bearing chemical groups called
CHROMOGENS
ABsorption
a PHYSICAL process by which the dye DISSOLVES
DIRECTLY INTO elements int he sample.
ADsorption
which the dye BINDS TO ELEMENTS IN THE SAMPLE that
have an affinity for the dye.
CONCENTRATION of the dye
the greater the concentration, the more the dye is bound to
tissue components
TEMPERATURE of the dye
increased temperature =increased rate of diffusion
throughout the tissue
pH of the staining sollution
cells have specific affinity for stains/dyes with specific pH
ranges
Tissue FIXATION
fixation alters and reorganizes certain molecular structures in
tissue samples so that they have an increased permeability
and are more receptive to staining.
T or F: Unfixed tissue elements have
LIMITED binding sites for dyes.
TRUE
pH scale: 1 - 14 1-6.9 ACIDIC. 7-7.4 NEUTRAL. 7.5-14 BASIC.
BUFFERS prevent fluctuations in pH
BASIC stains love ACID tissue
elements (nuclei & other basophilics)
ACID stains love BASIC tissue elements (cytoplasm & other
acidophilic)
OXIDATION =removal of electrons
from molecules
REDUCTION =adding electrons
in the case of staining, FIXATION
alters and reorganizes certain molecular structures in tissue
samples so that they have an increased permeability and are
more receptive to staining.
show positive PAS (Rose Red)
staining
Glycogen. Neutral mucins. Some epithelial mucins.
Basement membranes. Fungal walls.
PAS with Diastase
used in conjunction with the PAS staining procedure to
specifically identify glycogen granules in tissue samples.
Amylase enzymes in a malt extract are used to digest the
glycogen, which is then washed out of the tissue sample.
PAS counterstain with _______
hematoxylin
HARRIS hematoxylin with Acetic Acid
Mucicarmine
rose red dye in the mucicarmine staining solution gets its
color from the aluminum-carminic acid complex carmine
Mucicarmine results
Nuclei are stained black with Weigert's Iron Hematoxylin and
the remaining background tissue elements are stained yellow
with Metanil Yellow or Tartrazine.
Mucicarmine is also used to identify ADENOCARCINOMAS & Cryptococcus neoformans
Alcian Blue
has an affinity for acidic tissue elements like mucin. The dye
gets color from the copper in the molecule
Alcian Blue solution with a pH of 1.0
stains most SULFATED acid mucins. These mucins can be
found in cartilage, large intestine goblet cells and bronchial
serous glands.
Alcian Blue solution with a pH of 2.5
stains CARBOLYLATED mucins in connective tissues and
cartilage.
Alcian Blue used with PAS
both acid and neutral mucins can be demonstrated. Alcian
Blue will stain acidic mucins blue. PAS will stain neutral
mucins rose red. This technique is particularlyuseful in
diagnosing diseases of the gastrointestinal (GI) tract.
glutaraldehyde is s dialdehyde and
gives false result for PAS becuase
its 2nd NH2 group is left free to react w/Schiff (which is used
to detect aldehydes).
Glutaraldehyde fixes ____ and binds
________.
fixes slowly and binds slowly